Royal jelly supplemented soybean lecithin-based extenders improve post-thaw quality and incubation resilience of goat spermatozoa

The aim of the present study was to evaluate different concentrations of royal jelly (RJ) supplemented extenders for post-thaw quality and incubation resilience of goat spermatozoa. Semen samples were collected from five goats. Pooled semen were diluted with soybean lecithin-based extender without RJ (control) or supplemented with different concentrations (0.25, 0.5 and 0.75%) of RJ (RJ0.25, RJ0.5, RJ0.75 respectively), at a final concentration of 150 × 10⁶ spermatozoon/mL. Semen samples were assessed for sperm motility, plasma membrane integrity using hypoosmotic swelling test (HOST) damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL).The addition of RJ (0.5%, 0.75%) led to higher percentages of subjective motilities (55.33 ± 2.29%, 57.67 ± 2.58%) compared to control and RJ0.25 groups (49.00 ± 2,80%, 51.67 ± 3.09%) (P < 0.05) following the freeze–thawing process. RJ0.5 and RJ0.75 groups had higher plasma membrane functional integrities (66.40 ± 1.34%, 68.20 ± 2.05%) and lower defected acrosome rates (24.60 ± 3.36%, 23.80 ± 2.27%) compared to the other groups (P < 0.05). DNA damaged spermatozoa in all groups were not significant (P > 0.05).In the end of incubation, motility and HOST rates of RJ0.5 (14.00 ± 3.87%, 31.20 ± 3.70%) and RJ0.75 (15.00 ± 3.27%, 29.20 ± 2.59%) groups were higher than control (8.00 ± 2.54%, 18.20 ± 3.11%) and RJ0.25 (9.00 ± 2.07%, 20.60 ± 2.88%) groups (P < 0.05). Also defected acrosome and DNA fragmation rates of RJ0.5 (32.20 ± 1.30%, 5.4 ± 0.55%) and RJ0.75 (29.20 ± 1.30%, 5.80 ± 0.45%) groups were significantly lower than control (38.80 ± 0.84%, 7.40 ± 1.34%) and RJ0.25 (39.80 ± 2.05%, 7.00 ± 1.58) groups. This study shows that RJ supplemented extenders have beneficial effect on goat sperm parameters at 0 h and 6 h of incubation.     

Elamipretide as a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation

As a technique widely used in assisted reproduction, human spermatozoa cryopreservation makes it possible to conserve functional sperm for a long time, but the impact of cryodamage on sperm during the process could not be ignored. The objective of the present study was to investigate the efficacy of Elamipretide, a novel small mitochondrial targeting short cytoprotective peptide, in attenuating cryodamage during spermatozoa cryopreservation. Semen samples were collected and cryopreserved in freeze solution containing different concentrations (0.0, 0.1, 1, and 10 μM) of Elamipretide. Sperm motility, viability, membrane integrity, mitochondrial membrane potential, DNA fragmentation, antioxidant profiles, and acrosome reaction were measured and analyzed. The results showed that supplementation of the freeze media with Elamipretide (1 and 10 μM) significantly improved post-thaw sperm parameters including motility and viability, stability of the plasma membrane, and mitochondria and chromosomes. In addition, by adding Elamipretide, excessive oxidation and acrosome dysfunction in sperm cells undergoing freeze-thaw were also significantly attenuated. Therefore, Elamipretide may be a potential candidate for relieving cryodamage to human spermatozoa during cryopreservation.     

Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation

Ram sperm are subjected to extreme oxidative stress during their preservation at −196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4–5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance.     

Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures

Cryopreservation has become a central technology in many areas of clinical medicine, biotechnology, and species conservation within both plant and animal biology. Cryoprotective agents (CPAs) invariably play key roles in allowing cells to be processed for storage at deep cryogenic temperatures and to be recovered with high levels of appropriate functionality. As such, these CPA solutes possess a wide range of metabolic and biophysical effects that are both necessary for their modes of action, and potentially complicating for cell biological function. Early successes with cryopreservation were achieved by empirical methodology for choosing and applying CPAs. In recent decades, it has been possible to assemble objective information about CPA modes of action and to optimize their application to living systems, but there still remain significant gaps in our understanding. This review sets out the current status on the biological and chemical knowledge surrounding CPAs, and the conflicting effects of protection versus toxicity resulting from the use of these solutes, which are often required in molar concentrations, far exceeding levels found in normal metabolism. The biophysical properties of CPAs that allow them to facilitate different approaches to cryogenic storage, including vitrification, are highlighted. The topics are discussed with reference to the historical background of applying CPAs, and the relevance of cryoprotective solutes in natural freeze tolerant organisms. Improved cryopreservation success will be an essential step in many future areas such as regenerative medicine, seed banking, or stem cell technology. To achieve this, we will need to further improve our understanding of cryobiology, where better and safer CPAs will be key requirements.     

Effect of freezing and thawing on cell membranes of Lentinus edodes,the shiitake mushroom

When cell membranes of Lentinus edodes mycelium were rapidly frozen at either 50 or 160°C/min, viability was lost and this correlated with rupture of the plasmalemma and residual membrane material and with alterations in the organelles. Although with slow cooling (1°C/min) 80% of the samples recovered viability, some cells still showed similar changes to those cooled rapidly, indicating that individual cells of the mycelium do not respond in the same way.     

Effects of hypothermia on the survival and cryopreservation of minipig ileal cells and Chinese hamster ovary cells.

Temperature of culture can be used to modulate cellular metabolism for improving small intestinal cell culture and cryopreservation. An hypothermia pretreatment (2 days at 25°C and 3 hours recovery at 37°C) improved hamster cell survival to freeze-thaw damage (p < 0.01) but decreased the survival of 2 immortal pig ileal cell lines even though epithelioid IPI-2I cells were more tolerant to hypothermia than IPI-I fibroblasts. Epithelioid cells survived 3 days at 25°C with unaltered expression of cytokeratin-18 whereas colonies of fibroblasts did not survive more than a day at 25°C (p < 0.001). These results suggest that hypothermia-tolerance of pig ileal cell lines might differ according to cell lineage calling for further experiments on small intestinal primary cell culture.     

Effect of storage conditions on the LDL effectiveness in ovine sperm cryopreservation

The present study evaluated the effect of storage conditions on the LDL efficacy for cryopreserving ovine sperm. In this way, we compared egg yolk extender with three different forms for LDL storing, LDL diluted in Tris-glucose extender and stored in frozen (i) and freeze-dried (ii) states and LDL stored pure and added into the extender prior to use (iii). We also tested the effect of two storage temperatures (−20 and −80 °C) and three storage times (30, 60, 120 d). Frozen and freeze-dried extenders containing LDL, as well as LDL stored pure, improved post-thaw sperm quality. Storage temperatures did not influence negatively the cryoprotectiveness of LDL extenders. Furthermore, lyophilised LDL extenders stored at −20 °C were more effective in preserving sperm longevity than the other extenders stored at −20 °C. Finally, LDL extenders stored for 30 and 120 d were more efficient than 60 d in preserving ram sperm freezability.     

猪骨髓基质干细胞低温生物学渗透特性的研究

以猪为材料利用细胞计数仪法研究了骨髓基质干细胞的渗透特性,包括细胞的等渗体积、低渗或高渗溶液中细胞的平衡体积及细胞的不可渗体积.结果表明,在等渗条件下,骨髓基质干细胞的平均体积为5248.4μm~3,相当直径d为21.6μm;细胞体积随溶液渗透压的变化规律符合Boyle van’t Hoff关系式,据此得到猪骨髓基质干细胞的不可渗体积V_b=0.36Vi,为1902.5μm~3.     

Comparative cryobiological traits and requirements for gametes and gonadal tissues collected from wildlife species

A major challenge to retaining viability of frozen gametes and reproductive tissues is to understand and overcome species-specificities, especially because there is substantial diversity in cryobiological properties and requirements among cell types and tissues. Systematic studies can lead to successful post-thaw recovery, especially after determining: 1) membrane permeability to water and cryoprotectant, 2) cryoprotectant toxicity, 3) tolerance to osmotic changes, and 4) resistance to cooling and freezing temperatures. Although species-dependency ultimately dictates the ability of specific cells and tissues to survive freeze-thawing, there are commonalities between taxa that allow a protocol developed for one species to be useful information for another. This is the reason for performing comparative cryopreservation studies among diverse species. Our laboratory has compared cellular cryotolerance, especially in spermatozoa, in a diverse group of animals—from corals to elephants—for more than 30 yrs. Characterizing the biophysical traits of gametes and tissues is the most efficient way to develop successful storage and recovery protocols, but, such data are only available for a few laboratory, livestock, and fish species, with virtually all others (wild mammals, birds, reptiles, and amphibians) having gone unstudied. Nonetheless, when a rare animal unexpectedly dies, there is no time to understand the fundamentals of biophysics. In these emergencies, it is necessary to rely on experience and the best data from taxonomically-related species. Fortunately, there are some general similarities among most species, which, for example, allow adequate post-thaw viability. Regardless, there is a priority for more information on biophysical traits and freezing tolerance of distinctive biomaterials, especially for oocytes and gonadal tissues, and even for common, domesticated animals. Our colleague, Dr John Critser was a pioneer in cryobiology, earning that moniker because of his advocacy and devotion to understanding the differences (and similarities) among species to better store living genetic material.